Study On The Expression Of COL1A1,COL3A1,ELP2,STAT3 And P-STAT3 In AngⅡ-induced Myocardial Fibroblast Proliferation

Objective:In this paper,we intend to study the expression of type I collagen(Col1A1),type III collagen(Col3A1),elongation factor complex subunit 2(ELP2),signaling guide and transcriptional activator 3(STAT3),phosphorylated STAT3(p-STAT3)and the expression of various indexes after valsartan intervention in angiotensin II(Ang II)-induced myocardial fibroblast proliferation experiments.To investigate whether ELP2 affects the proliferation of Ang II-induced myocardial fibroblasts by regulating the expression of p-STAT3.Methods:The myocardial fibroblasts of C57BL/6 mice aged 1-3 days were isolated and purified by differential adherent trypsin digestion.The myocardial fibroblasts were identified by inverted microscope,and the primary cells were subcultured in the complete culture medium of myocardial fibroblasts.Taking the second generation of cardiac fibroblasts after passage,the experiment of Ang II stimulating the proliferation of cardiac fibroblasts and valsartan intervening the proliferation of cardiac fibroblasts was established.The experiment was divided into Control group(complete medium),Ang II0.5 group(Ang II 0.5umol/l),Ang II1.0group(Ang II 1.0umol/l)and Ang II1.0+ARB1.0 group(Ang II 1.0umol/l).After 24 hours of culture,CCK-8 experiment detected the absorbance value(OD value)and cell proliferation rate at 450 nm in each group,and Western Blot experiment(WB)detected the expression levels of proteins related to transcription regulation,proliferation and fibrosis in myocardial fibroblasts in each group.The expression levels of COL1A1,COL3A1,STAT3 and ELP2 m RNA in each group were measured by real-time quantitative PCR(rt-qpcr).Results:1.The myocardial fibroblasts of neonatal mice began to adhere to the wall at 1.5h,and basically adhered to the wall at 24 h.Microscopically,the cells were long and narrow,and grew dispersedly,which accorded with the characteristics of myocardial fibroblasts.2.CCK-8 results showed that compared with the control group,the OD value and value-added rate of Ang II 0.5 group and Ang II 1.0 group increased significantly(P < 0.05).Cell proliferation was dose-dependent with Ang II concentration(P < 0.05).After valsartan intervention,the OD value and cell proliferation rate decreased significantly compared with Ang II1.0 group(P < 0.05).3.Western blot results showed that the expression levels of COL3A1,STAT3,p-STAT3 and ELP2 protein in Ang II 1.0 group was significantly higher than that in the control group(P< 0.05).With the addition of Ang II concentration,the expressions of STAT3 and ELP2 protein increased significantly(P < 0.05),and the expression of COL1A1 protein also increased,but there was no significant difference(P > 0.05).After Valsartan treatment,the levels of COL3A1,STAT3,p-STAT3,ELP2 and other proteins decreased significantly(P < 0.05).4.RT-q PCR detection showed that the m RNA levels of COL1A1,COL3A1 and STAT3 in Ang II1.0 group were significantly higher than those in normal group,and there was significant difference between Ang II1.0 group and normal group.The expression of STAT3 protein increased with the increase of Ang II level,while that of Elp2 protein increased with the increase of Ang II level.Under the action of Valsartan,the expression of COL1A1 and STAT3 m RNA decreased significantly,and the expression of COL3A1 and ELP2 m RNA also decreased,but there was no significant difference between Ang II1.0 group and Ang II1.0+ARB1.0 group.Conclusion:(1)Ang II can induce the proliferation of myocardial fibroblasts and up-regulate the expression of myocardial fibrosis markers(COL1A1,COL3A1).And with the increase of Ang II concentration,the ability to stimulate the proliferation of myocardial fibroblasts is stronger.(2)Ang II may promote the proliferation of cardiac fibroblasts by activating STAT3 signal transduction pathway,and ELP2 may affect the proliferation of cardiac fibroblasts induced by Ang II by regulating the activation of STAT3.(3)Valsartan can prevent the proliferation of myocardial fibroblasts induced by Ang II to some extent,and down-regulate the expression of myocardial fibrosis markers(COL1A1,COL3A1).